Gostaríamos de agradecer o centro de genética Caenorhabditis para tensões, as ciências naturais e Conselho de pesquisa de engenharia (NSERC) e a Fundação do Canadá para a inovação (TPI) para financiamento. Gostaríamos de agradecer o Dr. Lizhen Chen (departamento de sistemas de célula e anatomia, UT saúde San Antonio) por permitir o uso irrestrito de seus laboratórios para todos os experimentos de c. elegans , bem como Dr. Exing Wang (diretor associado, Optical Imaging Facility UT saúde San Antonio) para obter assistência com microscopia confocal. Também gostaríamos de agradecer o Dr. Myron Ignatius para fornecer apoio e incentivo para facilitar a gravação do vídeo.

1. mancha e lisossomos intestinais de imagem
<ol><li>Placas de mídia de crescimento nematoide de semente (NGM) OP50
	<ol>
<li>Preparar pratos NGM conforme o protocolo recomendado19 e permitir que as placas fechadas secar por 2 dias à temperatura ambiente.</li>
		<li>Inocular as bactérias OP50 no caldo estéril de caldo Luria (LB) e crescer em um tremendo banho de incubadora ou água a 37 ° C por 36 h ou até que o OD está entre 0,2 e 0,4. Evitar o uso de uma cultura bacteriana com OD > 1 ou OD < 0.2.</li>
		<li>Uma vez que NGM placas são secas, coloque uma gota (~ 30 µ l) de inóculo OP50 no centro da placa e espalhe então a queda em um patch mais ou menos que é 2 x 2 cm de tamanho. Nota: Qualquer inóculo OP50 restante pode ser armazenado a 4 ° C por até um mês.</li>
		<li>Inverter as placas OP50, uma vez que o inóculo secou (cerca de 10 ou 15 min) e então incubar as placas a 37 ° C para 36 h.</li>
		<li>Após 36 h, retire as placas da incubadora e loja a 4 ° C até utilização posterior. Nota: As placas devem ser boas para até 1 mês a 4 ° C.</li>
	</ol></li>
	<li>Completando o cDCFDA para OP50
	<ol>
<li>Para complementar cDCFDA nas chapas OP50, prepare um estoque de trabalho de cDCFDA de 10 mM em dimetilsulfóxido (DMSO). Manter a solução de cDCFDA, protegida da luz e loja a-20 ° C para uso posterior.</li>
		<li>Coloque delicadamente a 100 µ l de solução de cDCFDA de 10mm para a superfície do patch OP50 2 cm x 2 cm, tal que o patch inteiro é uniformemente coberto com cDCFDA. Nota: É muito importante que o patch inteiro de OP50 é coberto com cDCFDA. Se necessário, use até 150 µ l de cDCFDA para cada placa. Também é imperativo que a solução de cDCFDA não se estende para além das fronteiras do patch OP50, desde que qualquer cDCFDA que flui fora o patch OP50 não ser incorporado a comida e resultará em intensidade de coloração reduzida.</li>
		<li>Colocar as placas de OP50 em uma caixa escura na parte superior do banco até que todos da solução cDCFDA é absorvido o patch bacteriano (geralmente cerca de 25 a 30 min).</li>
	</ol></li>
	<li>Coloração de vermes usando cDCFDA
	<ol>
<li>Uma vez que o cDCFDA completamente tem permeado o patch OP50, lugar não mais de 20 minhocas por placa e incubar as placas invertidas a 20 ° C por um período mínimo de 14 h. Nota: Recomenda-se colocar as placas de cDCFDA em um caixa opaco para minimizar a exposição à luz.</li>
		<li>Para controlar as diferenças de consumo entre experiências e para normalizar os sinais cDCFDA, (recomendado) co mancha com 3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato) ( diflúor) boro (2,5 µ l de uma solução de recém-feitos 1 mM), um corante de amina fraco acidotropic cuja fluorescência é em grande parte não-variante sobre o espectro de pH ácido (consulte Tabela de materiais para nomes comuns de reagentes). Nota: Não mancha worms para menos de 14 h, desde que este pode fornecer a intensidade de coloração inadequada cDCFDA e dar uma falsa interpretação de pH dos lisossomos.</li>
	</ol></li>
	<li>Preparar lâminas para microscopia
	<ol>
<li>Coloque duas tiras paralelas de rotulagem fita aproximadamente 3 polegadas separados sobre uma superfície lisa e plana, como um espelho (Figura 1).</li>
		<li>Revestir a superfície do espelho com spray repelente de água e limpe o líquido excedente.</li>
		<li>Derreter a agarose 2% (dissolvido em água destilada H2O) no microondas até completamente liquefeito, em seguida, coloque uma gota de µ l 50 de agarose entre as duas tiras de fita e rapidamente cobrir a gota com uma lâmina de microscópio, tal que o slide é perpendicular às tiras de fita. Depois que a agarose tem solidificado, formando uma almofada circular sob o slide, vire o slide e adicionar 10 a 15 µ l de 5mM de azida sódica (NaN3) sobre a placa de agarose. Nota: NaN3 é um inibidor do citocromo C que, quando usado em baixas concentrações, anestesia reversível vermes para que eles não vão se mover durante a imagem latente.</li>
		<li>Pegar vermes da placa OP50 suplementado com cDCFDA e transferi-los para um prato NGM limpo sem qualquer OP50. Permitir que as minhocas mover-se por alguns segundos permitir que a maior parte da OP50 para ser removido da superfície dos vermes e depois transferir os vermes para a almofada de agarose contendo azida sódica 5 mM.</li>
		<li>Cubra imediatamente o pad de agarose com uma lamela. Certifique-se de colocar suavemente a lamínula sem aplicar demasiada pressão, pois isso pode resultar em estouro de vermes. Nota: OP50 bactérias suplementadas com cDCFDA fluorescem quando visualizadas por microscopia, portanto, é importante limpar os vermes como o melhor possível antes de colocá-los na plataforma de agarose contendo azida de sódio. Se preparando mais de 6 cepas de c. elegans (incluindo N2, controle de tipo selvagem), é aconselhável preparar as amostras em lotes de 6, a fim de assegurar que os vermes não secar na rampa de agarose. Em algumas variedades, a vulva começa a romper-se após longos períodos de tempo devido à pressão da lamínula, portanto é importante planejar de acordo.</li>
	</ol></li>
	<li>Microscopia confocal Nota: Alguns microscópios têm um recurso interno que automaticamente aumenta a intensidade de fluorescência para compensar os níveis variáveis de fluorescência. Tais microscópios podem não funcionar bem para vermes manchados com cDCFDA, desde que eles inerentemente irão aumentar a intensidade de fluorescência das amostras de imagem.<ol>
<li>Executar a imagem em um microscópio confocal padrão usando comprimentos de onda de excitação/emissão definidos como 488/530 nm para cDCFDA. Leve o único avião imagens (não Z-stacks) de lisossomos intestinais e usar apenas os lisossomos que estão em foco (intensidade de sinal máxima) para quantificação de intensidade. Nota: Talvez devido a diferenças na disponibilidade de tintura, lisossomos das células intestinais diretamente posteriores da faringe mantêm cDCFDA coloração intensidade independentemente do genótipo ou idade, portanto, tenha cuidado para evitar a imagem de lisossomos nestas células.</li>
		<li>Imagem do slide que contém 2 dia de idade tipo selvagem que N2 vermes primeiro. Enquanto isso, ajuste os parâmetros da imagem latente confocal como potência do laser, pinhole e abertura para assegurar que a cDCFDA intensidade de coloração não é saturado. Nota: Uma vez que essas configurações são definidas, não alterá-los para toda a duração da imagem para esse dia.</li>
		<li>Capturar imagens de 1024 x 1024 pixel de um único avião do intestino (3 a 4 imagens por worm) usando um 60 X lente de ampliação. Nota: O espectro de emissão de cDCFDA em worms renderá um único pico proeminente em 520 nm coincidindo com sua fluorescência relatado espectro20, proporcionando uma leitura de sinal específico que é diferente de autofluorescência intestinal (Figura 3).</li>
		<li>Exporte cru confocal imagem (.lsm) como arquivos. TIFF para cada canal.</li>
		<li>Em seguida, abra o ImageJ e clique em arquivo | Abrir para abrir o arquivo de imagem para ser quantificada. Uma vez que carrega a imagem, use a ferramenta de seleção de forma oval no ImageJ para selecionar uma região de interesse (ROI).</li>
		<li>Depois disso, clique no Analyze | Medida para quantificar a intensidade da fluorescência para o ROI. Selecione 4 – 5 diferentes em foco lisossomos por imagem e calcular a intensidade de fluorescência relativa média de cada região de interesse (ROI).</li>
		<li>Para cada imagem, medir a intensidade de fluorescência de fundo em uma região do intestino entre lisossomos. Subtraia os valores de intensidade de fluorescência de plano de fundo dos valores de intensidade de fluorescência dos lisossomos.</li>
		<li>No total, colete em torno de 30 a 50 individuais valores normalizados para cDCFDA intensidade (animais de 6 a 10) para cada tensão a ser testado. Use esses valores para plotar uma caixa e whisker plot usando qualquer software estatístico adequado. Nota: A coloração pode variar consideravelmente dependendo de fatores como temperatura, tempo de incubação e a saúde geral/idade dos animais que comparações de intensidade de fluorescência só são relevantes no processo de amostras no mesmo experimento coloração. Portanto, é importante processar controles em cada experimento de coloração. Calcular as diferenças estatísticas (t-testes) usando qualquer software estatístico adequado.</li>
		<li>Imagem todas as cepas do mesmo experimento (manchado no mesmo dia) em sequência, tomando cuidado para não alterar nenhum dos parâmetros de imagem.</li>
	</ol></li>
</ol>

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Os autores declaram não haverá conflito de interesses.

Uma variedade de eventos celulares e moleculares contribuem para o envelhecimento, influenciado pela história de vida características e fatores genéticos. Nosso recente estudo22 sugere que o ciclo reprodutivo desempenha um papel importante no controle da aptidão da soma através da regulamentação da dinâmica dos lisossomos pH. Nós mostramos que a proteólise lisossomal mediada é promovido enquanto animais reproduzem-se ativamente por upregulation de transcrição v-ATPase, que por sua vez...

A aparência de agregados de proteínas é amplamente aceito para ser uma marca registrada de envelhecimento em pilhas eukaryotic1,2,3e a formação do que é pensado para estar entre os condutores de princípio da senescência celular4 , 5 , 6 , 7. há evidência crescente que, como células de idade catabolismo proteico é prejudicado, levando a um aumento na agregação da proteína. O colapso da proteólise em células de envelhecimento envolve uma imparidade de autofagia8 , bem como a degradação de proteínas mediada por Proteassoma9. Finalmente, oxidação de proteínas irreversível é aumentada em células velhas, prejudicando ainda mais o catabolismo de proteína10.
Autofagia foi inicialmente pensada para ser um processo não-seletivo para degradação em massa de proteínas danificadas, mas estudos recentes têm indicado que autofagia é altamente seletiva para o catabolismo de agregados de proteínas e organelas disfuncionais que não são passível de degradação através de de mecanismos de autorização outros proteína11. Durante o processo de autofagia, proteínas danificadas e agregadas são isoladas em uma vesícula de dupla membrana chamada o autophagosome. Este autophagosome então funde-se com as organelas ácidas chamadas lisossomos, o que leva à degradação do autophagosome carga12. Lisossomos representam o ponto de extremidade da via autophagic e participarem de diferentes processos celulares, tais como a reparação da membrana, controle transcricional e detecção de nutrientes; destacando seu papel centralizado na homeostase celular (revisto em ref. 13). Vários estudos têm demonstrado uma associação entre uma redução dependente da idade na função dos lisossomos e várias de doenças neurodegenerativas13. Consistentemente, restaurando a função dos lisossomos em células mais velhas pode retardar o aparecimento de fenótipos relacionadas ao envelhecimento14,15. Estudos da composição do meio de intralumen sugerem que o colapso da função dos lisossomos em células mais velhas não é devido a uma redução na produção de proteases lisossomais16. Como alternativa, tem sido proposto que perda da acidez intralysosomal, um requisito fundamental da sua actividade enzimática, pode fundamentam a diminuição da proteólise mediada por lisossoma17. Para ser capaz de explorar essa hipótese, é essencial desenvolver protocolos para sondar mudanças dinâmicas no pH dos lisossomos em células vivas de uma forma consistente e replicável e reagentes.
O intestino de c. elegans é o principal tecido metabólico em worms e é um crítico regulador da homeostase sistêmica e vida útil. Temos desenvolvido ensaios para avaliar alterações na acidez do lúmen de lisossomos intestinais de worms para determinar como mediada por lisossoma proteólise contribui para o envelhecimento. Embora sensíveis ao pH fluorophores têm sido utilizados anteriormente em c. elegans para marcar lisossomos intestinais, ainda não houve um esforço para estabelecer um protocolo bem sucedido que pode detectar pequenos aumentos no pH dos lisossomos na vivo18.. Aqui, nós fornecemos um protocolo que pode ser usado para detectar a perda da acidez dos lisossomos em células intestinais de c. elegans usando um protocolo de alimentação simples e conveniente que incorpora um fluoróforo sensíveis ao pH (cDCFDA) OP50 comida.

<table><tbody><tr><td>OP50 (&lt;em&gt;E. coli&lt;/em&gt;)</td><td>Caenorhabditis Genetics Center</td><td></td><td>Order online at https://cgc.umn.edu/strain/OP50</td></tr><tr><td>5(6)-carboxy-2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein diacetate&amp;nbsp;</td><td>ThermoFisher</td><td>C369</td><td>Commonly known as cDCFDA</td></tr><tr><td>9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#039;H-2,2&#039;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td><td>ThermoFisher</td><td>L7528</td><td>Commonly known as Lysotracker Red</td></tr><tr><td>Confocal microscope (e.g. Zeiss LSM 510)</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Download for free from https://imagej.nih.gov/ij/download.html</td></tr><tr><td>LB Broth powder</td><td>ThermoFisher</td><td>22700041</td><td></td></tr><tr><td>Bacto Agar</td><td>Sigma</td><td>A5306-1KG</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Bacto Peptone</td><td>Fisher Scientific</td><td>S71604</td><td></td></tr><tr><td>Cholesterol powder</td><td>Sigma</td><td>C3045&amp;nbsp;</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>449709</td><td></td></tr><tr><td>MgSO4</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>K3PO4</td><td>Sigma</td><td>P5629</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Cover Slips</td><td>ThermoFisher</td><td>3406</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A6013</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>Mirror or other smooth flat surface</td><td></td><td></td><td></td></tr></tbody></table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

O nemátodo Caenorhabditis elegans (c. elegans) é um sistema de modelo que é amplamente utilizado para estudar a longevidade e os caminhos do desenvolvimento. Tais estudos são facilitados pela transparência do animal, a capacidade de transmitir e reverter ensaios genéticos, a relativa facilidade de gerar proteínas fluorescente etiquetadas e o uso de corantes fluorescentes que podem também ser injetadas no início embrião ou incorporados em seu alimento (e. coli estirpe OP50) para rotular organelas celulares (por exemplo, 9-dietilamino-5h-benzo (a) phenoxazine-5-1 e (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1)) methylidene]-2 H-pyrrol-5-yl-kappaN} - N - [2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Aqui, apresentamos o uso de um corante fluorescente de sensíveis ao pH que manchas lisossomos intestinais, fornecendo uma leitura visual de alterações fisiológicas, dinâmicas na acidez dos lisossomos em worms ao vivo. Este protocolo não mede pH dos lisossomos, mas prefiro visa estabelecer um método confiável de avaliação fisiológicas relevantes variações na acidez dos lisossomos. cDCFDA é um célula-permeant composto que é convertido para o fluorescente fluoróforo 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) após hidrólise por esterases intracelulares. Protonação dentro de lisossomos armadilhas cDCF nestas organelas, onde se acumula. Devido a sua baixa pKa de 4.8, esse corante tem sido usado como um sensor de pH no fermento. Aqui nós descrevemos o uso de cDCFDA como suplemento alimentar para avaliar a acidez dos lisossomos intestinais em c. elegans. Esta técnica permite a detecção de lisossomos alcalinizante em animais vivos,, e tem uma ampla gama de aplicações experimentais, incluindo estudos sobre envelhecimento, autofagia e biogênese dos lisossomos.

cDCFDA manchas de lisossomos em uma maneira dependente do pH, e seu pKa baixo e captação pronta em lisossomos torna um sensor de pH ideal21. cDCFDA intensidade de coloração é inversamente proporcional dos lisossomos pH (ou seja, coloração aumentos de intensidade como pH diminui)18,22. os sinais cDCFDA são consistentemente fracos lisossomos de animais tratados com 20mm de cloroquina, um inib...

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Um guia passo a passo para sondar a perda da acidez dos lisossomos no intestino de c. elegans usando o corante vital sensíveis ao pH 6 - carboxi - 2', 7'-diclorofluoresceína diacetato (cDCFDA)

Avaliação dos lisossomos Alkalinization no intestino de viver elegans de Caenorhabditis

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

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Research

JoVE Journal

Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

5.9K visualizações.  University of Saskatchewan. Um guia passo a passo para sondar a perda da acidez dos lisossomos no intestino de c. elegans usando o corante vital sensíveis ao pH 6 - carboxi - 2', 7'-diclorofluoresceína diacetato (cDCFDA)

Vídeo: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Em pequena escala colorimétrica ensaios de intracelular lactato e piruvato no nemátode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Bioquímica

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

O c. elegans intestino como um modelo para intercelular Lumen morfogênese e In Vivo biogênese de membrana polarizada no nível de célula única: rotulagem por anticorpos, RNAi perda--função de análise e de imagem

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Biologia do Desenvolvimento

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Bioquímica e Alto Rendimento Avaliação microscópica de massa gorda em<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Biologia

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

Medindo os efeitos de bactérias e produtos químicos sobre a permeabilidade intestinal de Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

Medicina

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Avaliação da Dinâmica e Orientação em Caenorhabditis elegans Neurônios

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Neurociência

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Métodos para avaliar Subcelulares Compartimentos de músculo em<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Quantificação de lipídios abundância e avaliação da distribuição de lipídios em Caenorhabditis elegans por Nilo vermelho e coloração do óleo vermelho O

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imagem livre de rótulos da Dinâmica de Armazenamento Lipídido em Elegans caenorhabditis usando microscopia de dispersão raman estimulada

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

<meta charset="utf8"></head><body>Avaliação do metabolismo e do padrão de envelhecimento em Caenorhabditis elegans

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

<meta charset="utf8"></head><body>Autor em destaque: explorando processos metabólicos e de envelhecimento em C. elegans usando ensaios de baixo custo e alto impacto

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Usando corantes de baixo custo para visualizar o acúmulo de glicogênio e a integridade intestinal em Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

Avaliação dos lisossomos Alkalinization no intestino de viver elegans de Caenorhabditis

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